Lymphatic invasion is a major route for the spread of metastatic cancer cells. Yet despite the important role of lymphatics as a critical pathway for cancer metastasis, relatively little is known about tumor-associated lymphatic function, due in part to the inability to directly image the lymphatics in vivo. Recently, we demonstrated for the first time the ability to non-invasively and quantitatively image lymphatic contractile function in mice using dynamic near-infrared (NIR) fluorescence imaging with intradermal injection of a nonspecific NIR fluorophore, indocyanine green (ICG). Subsequently, the same approach was employed in mice with extensive lymph node (LN) metastasis to image alteration of lymphatic drainage pathways and lymphatic contractile activity. Based upon our initial findings, we propose to use non-invasive, dynamic functional NIR fluorescence imaging to longitudinally image tumor-associated lymphatics to test our working hypothesis that cancer metastasis is accompanied by transient changes of lymphatic contractile function and lymph flow patterns. Specifically, our aims are to: 1. Image spatial and temporal changes in lymphatic contractile function and drainage pathways longitudinally in response to tumor growth and LN metastasis in different experimental animal models of cancer in order to elucidate whether aberrant lymphatic function and lymph flow patterns are a prognostic marker of metastasis and, 2. Image spatial and temporal changes of the lymphatics in response to the anti-VEGF receptor (VEGFR)-32. Image spatial and temporal changes of the lymphatics in response to the anti-VEGF receptor (VEGFR)-3 blocking antibody or a combination of anti-VEGFR-2 and -3 blocking antibodies as a means to ameliorate the earliest tumor lymphangiogenesis and reduce the rate of metastasis in tumor-bearing mice. This project, if successful, could motivate translation of a new diagnostic for tumor-node- metastasis (TNM) staging and acceleration of clinical trials of emerging therapeutics that target the lymphatics. PUBLIC HEALTH RELEVANCE: Abnormal lymphatic function and flow are implicated in a number of pathological conditions, including cancer metastasis. Yet despite the important role of lymphatics as a critical pathway for cancer metastasis, relatively little is known about tumor-associated lymphatic function, due in part to the inability to directly image the lymphatics in vivo. In this work, we develop an approach to non-invasively and quantitatively image transient changes of lymphatic contractile function and flow patterns in response to tumor progression and metastasis using a near-infrared fluorescence imaging technique.